Precementum- and / or cementum - derived chemotactic factor (CCTF) of tooth of mammalia, process for purifying the same, and drug for accelerating adhesion of new connective tissue, comprising the same as active ingredient

ABSTRACT

A precementum- and/or cementum-derived chemotactic factor (CCTF) of a tooth of Mammalia, characterized in that a molecular weight measured by SDS-PAGE is 67000±1000.  
     A process for purifying a precementum- and/or cementum-derived chemotactic factor (CCTF) of a tooth of Mammalia, wherein a molecular weight measured by SDS-PAGE is 67000±1000, which comprises collecting precementum and/or cementum from an extracted tooth of Mammalia and immersing them in saline or collagenase-containing saline with stirring to obtain an eluted ingredient, and purifying the eluted ingredient by molecular weight fractionation, ion-exchange adsorption chromatography and hydroxyapatite adsorption chromatography.  
     A drug for accelerating adhesion of new connective tissue, comprising the precementum- and/or cementum-derived chemotactic factor (CCTF) as an active ingredient.  
     [FIG. 1] Gel filtration chromatography P: Positive control (10% FBS-containing DMEM liquid culture medium) A: Negative control (liquid culture medium alone) Fraction A, fraction B  
     [FIG. 2] DEAE-3SW ion-exchange chromatography P: Positive control: 10% FBS-containing DMEM liquid culture medium A: Negative control: DMEM liquid culture medium alone  
     [FIG. 3] Hydroxyapatitie chromatography P: Positive control: 10% FBS-containing DMEM liquid culture medium A: Negative control: DMEM liquid culture medium alone  
     [FIG. 4]  
     [FIG. 5] Western blotting of CCTF using anti-BSP-II antibody  
     [FIG. 6] Western blotting of CCTF using anti-BMP-2 antibody

TECHNICAL FIELD

[0001] The present invention relates to a precementum- and/orcementum-derived chemotactic factor (CCTF) of a tooth of Mammalia, aprocess for purifying the same, and a drug for accelerating adhesion ofnew connective tissue.

BACKGROUND ART

[0002] A periodontal disease is an inflammatory disease caused bybreakage of supporting tissue of teeth (e.g. gum, periodontal ligament,alveolar bone, etc.) due to bacterial plaque (cluster of bacteria), andthe periodontal disease and dental caries are referred to two typicaldiseases in the dental field.

[0003] In case of a severe periodontal disease, reconstruction of guidedtissue by a surgical technique is required. Its purpose lies in not onlyremoval of a periodontal pocket, but also restoring of an occludingfunction by regeneration of guided tissue broken due to the periodontaldisease. A current typical technique is a gingival flap operation,however, regarding a curing style after operation, true adhesion betweenthe tooth root and periodontal ligament (adhesion of new connectivetissue) attended with neogenesis of cementum is limited to the root apexand ideal adhesion is not recognized in the other portion (epithelialadhesion).

[0004] Under these circumstances, a trial of various techniques hasrecently been made to positively obtain adhesion of new connectivetissue. Among these techniques, a guided tissue regeneration technique(GTR technique) has attracted special interest. This GTR techniqueprevents or hinders the invasion of the defect portion of guided tissue,which has been curing, by gingival epithelium and gingival corium andthus allows the cells capable of attaching new connective tissue andforming an alveolar bone to grow into the defect portion, therebyregenerating guided tissue.

[0005] This technique is summarized as follows. First, a pocket formedby a periodontal disease is surgically removed and a gingival flap isdetached, and then polluted cementum corresponding to the pocket iscompletely scraped. The tooth root surface is covered with a membraneand a gum is sutured on the membrane.

[0006] To regenerate sound guided tissue according to the abovetechnique, it is necessary to induce migration, adhesion, growth anddifferentiation of gingival fibroblasts (hereinafter referred to ascells capable of adhering new connective tissue) to form newborncementum around the periphery of a tooth root, thereby to obtainadhesion of new connective tissue, and to form an alveolar bone.

[0007] However, according to a therapy for a periodontal disease whichis generally conducted at present, adhesion of new connective tissue andformation of an alveolar bone are insufficient.

[0008] An object of the present invention is to provide a substancewhich induces migration, adhesion, growth and differentiation of cellscapable of adhering new connective tissue, thereby to accelerateadhesion of the new connective tissue between the tooth and gum (toothroot and periodontal ligament) and to provide a process for isolatingand purifying the same, and to provide a drug for accelerating adhesionof new connective tissue, comprising the same as an active ingredient,which is used for restoring sound guided tissue with a periodontaldisease.

DISCLOSURE OF THE INVENTION

[0009] An object of the present invention is attained by a precementum-and/or cementum-derived chemotactic factor (CCTF) of a tooth ofMammalia, characterized in that a molecular weight measured by SDS-PAGEis 67000±1000.

[0010] An object of the present invention is also attained by a processfor purifying a precementum- and/or cementum-derived chemotactic factor(CCTF) of a tooth of Mammalia, wherein a molecular weight measured bySDS-PAGE is 67000±1000, which comprises collecting precementum and/orcementum from an extracted tooth of Mammalia and immersing them insaline or collagenase-containing saline with stirring to obtain aneluted ingredient, and purifying the eluted ingredient by molecularweight fractionation, ion-exchange adsorption chromatography andhydroxyapatite adsorption chromatography.

[0011] An object of the present invention is also attained by a drug foraccelerating adhesion of new connective tissue, comprising theprecementum- and/or cementum-derived chemotactic factor (CCTF) as anactive ingredient.

[0012] The present inventors have studied about already-known celladhesion and cell growth factors to solve the above problems, butbrought no good solution. Therefore, they have further studied and foundthat precementum and/or cementum of a tooth of Mammalia contains asubstance which has a chemotactic activity and causes invasion, growthand differentiation of cells capable of adhering new connective tissue,thereby to attract the cells to connective tissue.

[0013] The present inventors have also found that a novel chemotacticfactor (hereinafter referred to as CCTF) can be purified by immersingprecementum and/or cementum collected from extracted a tooth of Mammaliain saline or collagenase-containing saline with stirring to obtain aneluted ingredient, and subjecting the eluted ingredient to molecularweight fractionation, ion-exchange adsorption chromatography andhydroxyapatite adsorption chromatography. Thus, the present inventionhas been completed.

[0014] They have also found a drug for accelerating adhesion of newconnective tissue, comprising CCTF as an active ingredient, which isuseful for restoring sound guided tissue with a periodontal disease.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 is a graph showing an elution pattern obtained bysubjecting to HPLC gel filtration chromatography, after preparativecolumns TSK·G3000 TSK·G4000 for extract of precementum and cementum witha 1% collagenase-containing saline (pH 7) were connected each other inseries.

[0016]FIG. 2 is a graph showing an elution pattern obtained bysubjecting to DEAE ion-exchange chromatography, after mixingchromotactic activity fractions obtained in FIG. 1, each having amolecular weight of 270,000 to 290,000, 580,000 and 630,000 or more andconcentrating the mixture.

[0017]FIG. 3 is a graph showing an elution pattern obtained bysubjecting to HPLC hydroxyapatite chromatography, after concentrating achemotactic activity fraction (Fr. No. 13) obtained in FIG. 2 bydesalination.

[0018]FIG. 4 is a diagram showing an elution pattern obtained bymeasuring a chemotactic activity fraction (Fr. No. 14) CCTF obtained inFIG. 3 by SDS-PAGE (silver-colored portion is a detected portion).

[0019]FIG. 5 is a diagram showing a pattern of western blotting of CCTFusing anti-BSP-II antibody

[0020]FIG. 6 is a diagram showing a pattern of western blotting of CCTFusing anti-BMP-2 antibody.

BEST MODE FOR CARRYING OUT THE INVENTION

[0021] CCTF of the present invention is a glycoprotein whose molecularweight measured by SDS-PAGE is 67000±1000 which is obtained fromprecementum and/or cementum of a tooth of Mammalia. Also, CCTF has achemotactic activity and has an action of accelerating formation ofcells capable of adhering new connective tissue.

[0022] The fact that CCTF is not fibronectin (molecular weight: 450 Kd)and vitronectin (molecular weight: 76 Kd) as a chemotactic factor hasbeen proved by the fact that the chemotactic activity of CCTF was notinhibited by an antibody against fibronectin and vitronectin.

[0023] It has also been proved by western blotting that CCTF differsfrom BSP-II (molecular weight: 33.6 Kd, molecular weight of onecontaining a sugar chain: about 60 Kd) obtained by cementum which takespart in adhesion of gum cells and BMP-2 (molecular weight: 30 Kd) whichtakes part in regeneration of alveolar bone.

[0024] It has also been known that TGF-β and PDGF (platelet-derivedgrowth factor) take part in regeneration of bone, in addition to BSP-IIand BMP-2. It has been found that CCTF is not TGF-β or PDGF because thechemotactic activity of CCTF is not inhibited by these antibodies, i.e.anti-TGF-β antibody and anti-PDGF antibody, thereby making it clear thatthere is no substance corresponding to cell adhesion factors and cellgrowth factors which have ever been reported.

[0025] To obtain CCTF of the present invention, teeth extracted fromMammalia are used. Among Mammalia, bovine teeth are preferably usedbecause of large population (a lot of bovines are now breeding for thepurpose of using for food), easy availability of teeth, and large amountof CCTF obtained from a tooth.

[0026] CCTF is purified in the following procedure.

[0027] After periodontal ligament fibers are removed from an extractedtooth by using a scaler, precementum and/or cementum are scraped byusing a sharp scaler and dispersed in saline (pH 7.0±0.5) containing0-5% collagenase (collagenase is not contained in case of using onlyprecementum), and then a protein ingredient is eluted with stirring. Aninsoluble matter is removed from the eluate by centrifugal separation.The eluate is subjected to membrane filter filtration and the filtrateis subjected to molecular weight fractionation such as gel filtration orultrafiltration to obtain a fraction having a molecular weight of270,000 or more.

[0028] The fraction is washed with a 50±5 mM tris ethanolaminehydrochloride buffer by DEAE ion-exchange chromatography (afteradsorbing on a resin at pH of 7.5±0.5), and then a fraction to be elutedwith a 50±5 mM tris ethanolamine hydrochloride buffer containing 0.2-0.3M sodium chloride is collected and concentrated by desalination.

[0029] The concentrated solution is adsorbed on hydroxyapatiteequilibrated with a 10±1 mM phosphate buffer (pH 6.6±0.3 mM) and, afterwashing with a 10±1 mM phosphate buffer (pH 6.6±0.3 mM), the fraction tobe eluted with 80-110 mM phosphoric acid is collected.

[0030] The substance thus obtained exhibits a single band of 67±1 Kd bySDS-PAGE. The substance is a glycoprotein containing sugar chains havingan amino acid composition of: Asp; 10.6±0.5%, Thr; 3.7±0.3%, Ser;13.3±0.7%, Glu; 13.8±0.7%, Gly; 23.3±1.2%, Ala; 10.1±0.5%, Cys/2;3.6±0.3%, Val; 6.7±0.3%, Ile; 3.8±0.3%, Leu; 7.3±0.4% and Lys; 3.8±0.3%.The isoelectric point is 6.5±0.5.

[0031] The glycoprotein thus obtained is a substance which has achemotactic activity and also has an action of accelerating adhesion ofnew connective tissue.

[0032] When using CCTF thus obtained of the present invention as a drugfor accelerating adhesion of new connective tissue, for example, thoseobtained by immersing a collagen membrane, a gelatin membrane or otherbiodegradable membrane with the drug are applied to the affected part.Alternatively, the drug is applied in the form of paint, or applied bysubcutaneous injection.

[0033] Specific direction for use includes, for example, process ofaccelerating adhesion of new connective tissue after operation byinserting those, which are obtained by immersing a collagen membrane, agelatin membrane or other biodegradable membrane with a chemotacticfactor of the present invention, into space between the tooth rootsurface smoothened by scraping a pollutant and the gum when applied tothe flap operation as a therapy for periodontal disease. Sound guidedtissue can be restored by using CCTF in such a manner.

EXAMPLES

[0034] The following Examples further illustrate the present inventionin detail.

Example 1

[0035] First, periodontal ligament fibers of forty bovine teethextracted from butchered bovines were removed not remaining using ascaler. Then, cementum containing precementum was scraped using a sharpscaler and dispersed in 20 ml of saline (pH 7) containing 1%collagenase. After the dispersion was stirred using Voltex (AutomaticMixer S-100, manufactured by Taiteck Co.) for 10 minutes, a proteiningredient was eluted. Furthermore, the insoluble matter was removed bycentrifugal separation at 3000 rpm for 5 minutes to obtain asupernatant. Then, the supernatant was filtrated through a membranefilter having a pore diameter of 0.22 μ (Durapore, manufactured byNippon Milli pore Co.) and the filtrate was concentrated to 4 ml (11 mgof a protein content measured by Bradford's process). Preparativecolumns TSK G3000 (2.15 cm in inner diameter×60 cm in length,manufactured by Toso Co.) and TSK G4000 (2.15 cm in inner diameter×30 cmin length, manufactured by Toso Co.) were connected each other inseries, and then molecular weight fractionation was conducted by HPLCgel filtration chromatography.

[0036] Each 4.2 ml of a fraction was collected and active fraction wasdetermined in accordance with chemotaxis assay [K W. Falk et al. ,Immunol. Methods, Vol.33, 239-247 (1980)] using human gingivalfibroblasts.

[0037] Specifically, a D-MEM (serum free) medium containing 10% of eachmolecular weight fraction was divided into bottom wells in the chamberat 25 μl of medium/well using a 96-well microchemotaxis chamber (NeuroProbe, Cabin John, Md., USA). Furthermore, a D-MEM (10% FBS) medium wasused as a positive control, while a D-MEM (serum free) was used as anegative control. On the bottom chamber, a filter having a pore diameterof 8 μm, a rubber and a top chamber were placed and fixed in thissequence. The D-MEM medium containing human gingival fibroblasts of5×10⁵ cells/ml was divided into wells in the top chamber at 50 μl ofmedium/well. The chamber was incubated for 3 hours at 37° C. under 5%CO₂-95% air. After the completion of the incubation, cells were fixedand stained with a Diff-Quich staining solution (International Reagents& Co., Japan), washed with water and then air-dried. The number of cellswhich migrated to the lower surface of the membrane filter were counted.The number of cells in a high power field was counted for each well byvisually observing using an optical microscope (objective lens;×20)wherein a grid micrometer is inserted into an ocular lens (×10).

[0038] As a result, a chemotaxis activity was recognized at fractions 40to 41 having a molecular weight of about 580,000 or more (hereinafterreferred to as “faction A”) and fractions 47 to 49 having a molecularweight of about 270,000 to 290,000 (hereinafter referred to as “factionB”). The results are shown in FIG. 1.

[0039] Fractions A and B did not exhibit activity corresponding to TGF-βor BSP-II, and they were not deactivated by treating at 60° C. for 30minutes.

[0040] It has already been found by a preparatory experiment thatfractions A and B exhibit the same molecular weight band by SDS-PAGEwhen purified by DEAE ion-exchange chromatography and hydroxyapatitechromatography. Therefore, they were combined, concentrated byultrafiltration to 2 ml. Then, the concentrate was adsorbed on a DEAEion-exchange resin (TSK, DEAE-3SW, manufactured by Toso Co.) at pH 7.5,subjected to DEAE ion-exchange chromatography (DEAE-3SW: 7.5 mm in innerdiameter×7.5 cm in length, manufactured by Toso Co.), washed with a 50mM tris ethanolamine hydrochloride buffer and eluted with a gradient ofa 0-0.5 M sodium chloride-containing 50 mM tris ethanolaminehydrochloride buffer. Furthermore, each 1 ml of fraction was collectedand the active fraction was determined by chemotaxis assay describedabove. As a result, the activity was recognized at the fraction elutedwith a 3M sodium chloride-containing 50 mM tris ethanolamine buffer. Theresults are shown in FIG. 2.

[0041] Then, each active fraction was concentrated by desalination. Theconcentrated fraction was subjected to chromatography on hydroxyapatiteequilibrated with a 10 mM phosphate buffer (pH 6.8) (10 m in innerdiameter×10 cm in length, manufactured by Koken Co.), washed with a 10mM phosphate buffer (pH 6.8) and eluted with a gradient of 0-200 mMphosphate. Furthermore, each 1.5 ml of fraction was collected and theactive fraction was determined by chemotaxis assay. As a result,chemotaxis activity was recognized in the fraction eluted with 80-110 mMphosphate. The results are shown in FIG. 3.

[0042] The fraction was concentrated by desalination to obtain 700 ng ofCCTF. CCTF thus obtained exhibited a single band of 67±1 Kd by SDS-PAGE.The results are shown in FIG. 4.

[0043] Furthermore, this band was not stained by western blotting usinganti-BSP-II antibody and anti-BMP-2 antibody. Thus, it has been foundthat CCTF is a substance different from BSP-II and BMP-2 (see FIGS. 5and 6).

[0044] CCTF thus obtained has an amino acid composition of: Asp; 10/6%,Thr; 3.7%, Ser; 13.3%, Glu;13.8%, Gly; 23.3%, Ala; 10.1%, Cys/2;3.6%,Val; 6.7%, Ile; 3.8%, Leu; 7.3% and Lys; 3.8%, and was an glycoproteincontaining sugar chains. The isoelectric point was 6.5±0.5.

[0045] Then, 4 ml of a 5% collagen solution (type I-A) was prepared. Tothe solution, 200 ng of CCTF was added. 0.4 ml of the mixture wasdivided into each well in a 24-well plate (a well: bottom area; 1.77cm², height; 2 cm) and polymerized in an open system. After 30 minutes,5×10⁴ of human gingival fibroblasts were inoculated on the collagen geland then cultured for 3 days at 37° C. in the presence of 5% carbondioxide. A sample for electron microscopy was prepared, embedded inepon, and then stained with toluidine blue. The invasion distance intoCCTF-containing collagen and the number of inoculated human gingivalfibroblasts were determined by visually observing using an opticalmicroscope.

[0046] Furthermore, the effect of non-addition, or addition offibronectin, TGF-β, PDGF, or CCTF and anti-vitronectin antibody wasexamined in the same manner as described above. The results are shown inTable 1. TABLE 1 Number of Invasion invaded Distance No. cells (μm) 1**No addition 3.6  62.1 2* CCTF 200 ng 8.3 136.4 3** TGF-β 200 ng 5.5102.0 4** Fibronectin 200 ng 4.8  72.3 5** PDGF 200 ng 7.1 122.4 6*CCTF(200 ng) + 8.1 136.2 anti-vitronectin

[0047] As shown in Table 1, in case where CCTF are added, both of thenumber of invaded cells and invasion distance into collagen gel wassignificantly higher than in case of no addition or addition offibronectin, TGF or PDGF. The action of CCTF was not inhibited byanti-vitronectin antibody.

Example 2

[0048] First, periodontal ligament fibers of forty bovine teethextracted from butchered bovines were removed not remaining using ascaler. Then, cementum and precementum were scraped using a sharp scalerand dispersed in 20 ml of saline (pH 7) containing 1% collagenase. Afterthe dispersion was stirred using Voltex (Automatic Mixer S-100,manufactured by Taiteck. Co.) for 10 minutes, a protein ingredient waseluted. Furthermore, an insoluble matter was removed by centrifugalseparation at 3000 rpm for 5 minutes to obtain a supernatant. Then, thesupernatant was filtrated through a membrane filter having a porediameter of 0.45 μ (Durapore, manufactured by Nippon Milli pore Co.) andthe filtrate was concentrated to 4 ml using an ultrafilteration membranehaving a fractionated molecular weight is 200,000.

[0049] Then, the concentrate was adsorbed on a DEAE ion-exchange resin(TSK, DEAE-3SW, manufactured by Toso Co.) at pH 7.5, subjected to DEAEion-exchange chromatography (DEAE-3SW: 7.5 mm in inner diameter ×7.5 cmin length, manufactured by Toso Co.) and eluted with a gradient of a0-0.5 M sodium chloride-containing 50 mM tris ethanolamine hydrochloridebuffer. The fraction eluted with a 0.2-0.3 M sodium chloride-containing50 mM tris ethanolamine hydrochloride was collected and thenconcentrated by desalination.

[0050] The concentrated fraction was subjected to chromatography onhydroxyapatite equilibrated with a 10 mM phosphate buffer (pH 6.6) (10mm in inner diameter×10 cm in length, manufactured by Koken Co.) andthen eluted with a gradient of 0-200 mM phosphate. The fraction elutedwith 80-110 mM phosphate was collected and concentrated by desalinationto obtain 730 ng of CCTF. CCTF thus obtained exhibited a single band of67±1 Kd by SDS-PAGE.

[0051] Furthermore, this band was not stained by western blotting usinganti-BSP-II antibody and anti-BMP-2 antibody. Thus, it has been foundthat CCTF is a substance different from BSP-II and BMP-2.

[0052] Then, 10 ml of a 5% collagen solution (type I-A) was prepared andthe solution was mixed with 600 ng of CCTF described above. The mixturewas poured in a thick of 2 mm and freeze-dried to form a CCTF-containingcollagen membrane. This was subjected to sterilized by ultraviolet lightfor two hours immediately before use.

[0053] After gingival flap of left and right incisors and lateralincisors of an upper jaw of a monkey was exfoliated and tumbled on theside of a labellum, an alveolar bone was cut out toward the side of anapiculus by about 4 mm to expose a tooth root. The exposed tooth rootwas smoothened by scraping cementum on the surface. The exposed lefttooth root face was covered with CCTF-containing collagen membrane,while the exposed right tooth root face was left as it was, and then thegingival flap was replaced and sutured. After operation, plaque controlwas applied everyday for three weeks until curing is recognized.

[0054] After three weeks, the animal was butchered, perfused and fixed.A tissue block was collected to prepare a paraffin section and then H-Estaining was applied. Histological observation revealed that adhesion ofnew connective tissue has been recognized in the left one treated withCCTF-containing collagen. Formation of an alveolar bone has also beenrecognized. On the other hand, both of them has not been recognized inthe right one.

Industrial Applicability

[0055] The invention of claim 1 provides CCTF which accelerates adhesionof new connective tissue by applying to the defect portion, thereby torestore sound guided tissue.

[0056] According to the purification process of the invention of claim4, CCTF of the present invention can be efficiently extracted andpurified.

[0057] According to the invention of claim 7, there can be provided adrug for accelerating adhesion of new connective tissue between thetooth and gum (tooth root and periodontal ligament).

1. A precementum- and/or cementum-derived chemotactic factor (CCTF) of a tooth of Mammalia, characterized in that a molecular weight measured by SDS-PAGE is 67000±1000.
 2. The chemotactic factor (CCTF) according to claim 1, which is a glycoprotein containing sugar chains having an amino acid composition of: Asp; 10.6±0.5%, Thr; 3.7±0.3%, Ser; 13.3±0.7%, Glu; 13.8±0.7%, Gly; 23.3±1.2%, Ala; 10.1±0.5%, Cys/2; 3.6±0.3%, Val; 6.7±0.3%, Ile; 3.8±0.3%, Leu; 7.3±0.4% and Lys; 3.8±0.3%, and has an isoelectric point of 6.5±0.5.
 3. The chemotactic factor (CCTF) according to claim 1 or 2, wherein Mammalia is bovine.
 4. A process for purifying a precementum- and/or cementum-derived chemotactic factor (CCTF) of a tooth of Mammalia, which comprises eluting a protein ingredient from precementum and/or cementum of a tooth of Mammalia and purifying the precementum- and/or cementum-derived chemotactic factor (CCTF) of a tooth of Mammalia by molecular weight fractionation, ion-exchange adsorption chromatography and hydroxyapatite adsorption chromatography.
 5. A process for purifying the chemotactic factor (CCTF) of claim 4, which comprises: (a) collecting precementum and/or cementum from an extracted tooth of Mammalia and immersing them in saline or collagenase-containing saline with stirring, thereby to elute a protein ingredient; (b) removing an insoluble matter from the eluate in the step (a) by centrifugal separation and filtration, and subjecting the resulting filtrate to a process such as gel filtration or ultrafiltration to obtain a fraction having a molecular weight of 270,000 or more; (c) adsorbing the fraction obtained in the step (b) on a DEAE ion-exchange resin, collecting a fraction eluted with a 0.2-0.3 mM sodium chloride-containing tris ethanolamine hydrochloride buffer, and concentrating the fraction by desalination; and (d) adsorbing the concentrated solution obtained in the step (c) on hydroxyapatite equilibrated with a phosphate buffer, and collecting the fraction to be eluted with 80-110 mM phosphoric acid.
 6. The process for purifying the chemotactic factor (CCTF) according to claim 4 or 5, wherein Mammalia is bovine.
 7. A drug for accelerating adhesion of new connective tissue, comprising the precementum- and/or cementum-derived chemotactic factor (CCTF) of any one of claims 1 to 3 as an active ingredient. 